Evolutionary pressure has favored mechanisms that allow the body to efficiently store nutrients as fat when food is abundant as a safeguard against occasional famine. With the dramatic changes in modern lifestyle including consumption of high carbohydrate and high fat foods, these mechanisms may now be contributing to a major epidemic of obesity in the US where the majority of the population is overweight. Glucose is not only a major fuel of all mammalian tissues but also a source of carbon for fat and protein synthesis. The liver is the principal organ responsible for the conversion of excess dietary carbohydrate into triglycerides. Ingestion of a high carbohydrate diet induces transcription of more than 15 genes involve in the conversion of glucose to storage fat. A transcription factor, purified from rat liver, with specificity for carbohydrate responsive elements (ChRE) found in the promoters of multiple genes required for lipogenesis and which displayed appropriate dietary responsive regulation was first identified in this laboratory and termed "carbohydrate response element binding protein, ChREBP". The complete process by which ChREBP is activated in response to excess carbohydrate in order to induce the transcription of lipogenesis enzyme genes, and then is turned off, is not yet fully understood. We have shown that glucose and cAMP have opposing activities in the regulation of lipogenesis, in part through dephosphorylation/phosphorylation of multiple sites on ChREBP. Glucose stimulates dephosphorylation of at least some of these sites by activating a Xu5P-stimulated protein phosphatase, Xu5P-PP2A. We recently found that the interaction of ChREBP with 14-3-3 is one of the most important steps regulating the nuclear localization of ChREBP. Phosphorylation of ChREBP activates its binding to 14-3-3 and is essential for ChREBP export out of the nucleus under starvation conditions. In addition, we found more recently that a specific metabolite in liver promotes the interaction of ChREBP with 14-3-3. To characterize the metabolite further we propose to (1) isolate the metabolite in pure form and determine the structure, (2) determine the mechanism by which it activates the interaction between ChREBP and 14-3-3, (3) determine how the metabolite is synthesized and degraded, what enzymes are responsible for these reactions and how they are regulated, (4) investigate whether the metabolite might be a signaling compound for regulation of the subcellular localization of ChREBP in response to nutrients and starvation, and (5) elucidate how Xu5P activates ChREBP and the bifunctional enzyme, Fru6P, 2kinase/Fru2,6 bisPase, using X-ray crystallography. PUBLIC HEALTH RELEVANCE: Obesity and its associated diseases, diabetes, hypertension, heart disease and some cancers are among the most serious health problems now facing the US. The efficient conversion of excess carbohydrates to fat for long-term storage, which until modern times it would appear was an evolutionary advantage in adapting to uncertain food supplies, now appears to contribute significantly to development of obesity. Carbohydrate response element binding protein (ChREBP) is a transcription factor that becomes active when carbohydrates are eaten and increases the expression of genes required for synthesis of fatty acids. The results of the work proposed in this application will elucidate the mechanisms involved both in activation and termination of ChREBP dependent increases in enzymes needed to make fatty acids. This knowledge will provide information that may be useful in developing strategies for the prevention and treatment of obesity.